Exemplary aspects of the present invention relate to a method to immobilize nucleic acids, a method to manufacture a biosensor using this method, and a method to detect nucleic acids.
A biosensor is being used to detect a target molecule in a sample by taking advantage of unique interactions of biorelated molecules, such as enzyme-substrate reaction, antigen-antibody reaction, and hybridization of nucleic acids in basic research, blood test and genetic analysis in the field of medicine, process control in food industry, and environmental measurement.
The biosensor includes a biorelated substance as a sensing part that reacts uniquely with a target substance and a converter which converts changes caused by interactions between the target substance and the sensing part into physical signals, such as electric current or voltage. Accordingly, in order to make a highly sensitive biosensor, it is necessary to optimize the sensing part structure and a reading device to read the signals.
One way to immobilize nucleic acids as the sensing part of the biosensor is to form a thin film containing nucleic acids on a surface of a solid support, such as a substrate. In order to form the thin film, a linker is put between ends of the nucleic acid molecules in advance to bond with specific groups so that the molecules are absorbed onto the surface of the solid support. The specific group is a thiol group, for example. By first forming a gold thin film on the solid support surface, a self-assembled monolayer (SAM) of a nucleic acid probe is formed on the solid support surface through gold-sulfur bond. Thus, the sensor on which a DNA probe, having a well-known base sequence, is immobilized and which detects hybridization of this probe with the nucleic acids contained in the sample by using physio-chemical signals (e.g., volume of fluorescence) is now in practical use.
The efficiency in the hybridization of the target nucleic acid contained in the sample with the DNA probe immobilized on the biosensor largely depends on densities of the DNA probes (See Peterson, A. W. et al., Nucleic Acids Research, vol. 29, No. 24, 5163-5168 (2001)). In order to enhance sensitivity of the DNA sensor, it is necessary to maximize the densities of evenly dispersed DNA probes while maintaining distance between the probes as required for hybridization. To control the immobilizing DNA probes so as to have optimal densities, a method in which a relatively low-volume molecule called a spacer molecule is suitably inserted between the nucleic acid probes is employed, for example. There is a report on an experiment in that the probe is a single-stranded DNA with a thiolated end and that 6-mercapto-1-hexanole is used as the spacer molecule (See Tonya M. Herne et al., Journal of the American Chemical Society, 119, pp. 8916-8920 (1997)).
One of the methods generally employed upon using the spacer molecules is a method in which the DNA probes are first absorbed onto the substrate and, thereafter, the spacer molecules are embedded therein. Another such method is coabsorption in that a mixed solution of the DNA probes and spacer molecules is immobilized in one step. Mainly employed at present is the former. However, the process to form the film through various steps requires a relatively long time, and fine adjustment of the densities of the DNA probes is not without difficulties.
With the coadsorption method, it is possible to save process time because this film formation is performed by one step. At the initial stage, by merely adjusting composition ratios of various kinds of molecules including the DNA probes in the solution at the initial stage, it is logically possible to adjust the densities of the nucleic acid probes on the biosensor by using the method in that the solid support is immersed in this mixed solution and that the volumes of the molecules absorbed on the solid substrate are adjusted by altering mixing ratios of these molecules.